IRON-DEPENDENCE OF CLOFAZIMINE ACTIVITY IN MYCOBACTERIA ABSTRACT Clofazimine (CFZ) is a riminophenazine dye that can be highly bactericidal against mycobacteria. Originally developed for use against Mycobacterium tuberculosis (Mtb), it has been primarily used for the treatment of leprosy and is listed by the WHO as an Essential Medicine for adults and children. CFZ has recently been included in the WHO recommended five drug short-course for MDR-TB. However, the efficacy of CFZ for the treatment of Mtb infections in animal models and clinical trials has been mixed. Historically, CFZ demonstrated high activity against Mtb in vitro and in murine models; however, CFZ efficacy was low in other animal models. A recent report indicates that differences in CFZ activity are based on the microenvironment. In a standard BALB/c mouse model CFZ was highly active, whereas in the ?Kramnik? mouse model, CFZ failed to demonstrate activity against Mtb if administered after a point at which necrotic lesions had already formed. The primary difference between models in which CFZ is active and those in which it is not appears to be that necrotic granulomas exist in models in which CFZ is less active. CFZ is a redox-active molecule that interacts with the NADH reductase II of the electron transport system. Mutations that increase expression of MmpL5 confer moderate resistance to CFZ. MmpL5 belongs to the RND efflux family and is important in the export of the mycobacterial siderophores; membrane-bound mycobactin and the soluble form carboxymycobactin. Our data indicate that CFZ bactericidal activity is dependent on iron availability and the presence of the mycobactin iron acquisition system. We have also demonstrated that CFZ exposure induces iron acquisition genes and most importantly CFZ drains Mtb intracellular iron levels indicating CFZ acts via the depletion of intracellular iron. The goals of this project are to determine if CFZ kills through intracellular iron depletion and determine if CFZ efficacy can be improved in disease microenvironments, where iron is readily available, by inhibiting Mtb iron acquisition mechanisms. To achieve these goals we will determine the role of iron availability, acquisition mechanisms, and storage on CFZ killing and intracellular iron levels. We will also determine if chemical inhibition of Mtb iron acquisition will synergize with CFZ to allow lethal activity in necrotic lesions of the Kramnik mouse model.